Cell fate establishment during early development of cyprinid fishes, with special emphasis on the formation of the primordial germ cells.

Cell fates can be established either by preformation or by epigenesis. With respect to primordial germ cells (PGCs) it has been shown that the Amphibia exhibit both types of cell fate establishment. Therefore, it is important to study the germ cell origin of the evolutionary lower class of fishes.In order to study the origin and cell fate establishment of a specific cell type, two approaches are possible. Firstly, its morphological characteristics may be studied, and traced back to the earliest possible developmental stage. Using this approach, we studied in Barbus conchonius, a cyprinid fish, the presence, location and morphology of cells, containing nuage, an ultrastructural electron dense perinuclear material, generally accepted to be characteristic for germ cells (Chapter 2). The results show that nuage containing cells, PGCs, could be found from 10 h after fertilization (a.f.) onwards (around 100% epiboly). They translocate between 10 h and 12 h a.f. from a position within the mesoderm towards a position between mesoderm and yolk syncytial layer (YSL). However, PGCs remain separated from the YSL by extensions of endodermal cells. The location of PGCs at the stage of first identification may indicate that they originate within the mesoderm. Contacts with endodermal cells may be involved in the formation of their characteristics.A second approach for studying the origin of PGCs concerns the labeling of blastomeres during cleavage stages and following their progeny up to the stage of morphological recognition of PGCs (Chapter 3). Using this method, a cell lineage tracer, Lucifer Yellow - Dextran (LY-D), was injected into individual blastomeres of the 64 cell stage of Barbus conchonius embryos. Study of the fate of their progeny revealed that a lower layer cell (LLC) could give rise to both somatic cells and a number of PGCs (in about 25% of the cases), while injections into upper layer cells (ULC) only resulted in labeled somatic cells. The distribution of somatic progeny after injection of a certain blastomere was unpredictable with respect to both the later location within the embryo and the tissue type of its descendants. Unpredictable cell lineages were probably due to extensive intermingling of cells during epiboly. The results suggest that the formation of PGCs as well as that of somatic cells is an epigenetic process. As mentioned above, the PGCs may originate within the mesoderm (Chapter 2).Since in Barbus conchonius cells apparently are not yet committed to their fate during cleavage and start extensive intermingling during epiboly, the question arose when cell fate restrictions occur (Chapter 4). These restrictions may also be important for the determination of a diversity of cell types, including PGCs. Changes in communication properties between cells or cell groups may be correlated with differences of their developmental pathways. Since it was known that during early epiboly certain deep cells are dye-coupled to the yolk syncytial layer (YSL), injections of Lucifer Yellow (LY) into the YSL at several stages of epiboly were used in order to study changes in communication properties. The formation of a group of dye-coupled cells, indicating a group of gap junctionally communicating cells, was described. At the onset of epiboly LY appeared to be transferred from the YSL to all blastodermal cells. Between 40% and 60% epiboly we observed a ringshaped group of labeled cells, which probably had involuted during early gastrulation. This cell group correlated with the leading edge of the hypoblast, and was dye-uncoupled from the uninvoluted epiblast. From 60% epiboly onwards the blastodermal cells were dye-uncoupled from the YSL. Between 50% and 100% epiboly the ring-shaped labeled hypoblast was, due to convergent cell movements, gradually transferred towards a dorsally located bar-like structure. Gap junctions appear to connect cells with the same fate. Consequently, the appearance of part of the hypoblast as a dye-coupled cell layer, which is dye-uncoupled from the epiblast, may correlate with early restriction in cell fate. On the other hand, gap junctions between YSL and DC may be involved in the transfer of an inducing signal at the onset of gastrulation.Apparently, it is only from gastrulation onwards that developmental pathways of cell groups gradually disperse. Since cell determination appeared to be highly regulative, knowledge of the control of the directional mass cell migration during gastrula stages may be important for understanding cell fate establishment. Because fibronectin (FN) appeared to be involved in the guidance of migrating cells in amphibian embryos, the presence of FN during epiboly and gastrulation was studied in embryos of the common carp ( Cyprinus carpio ; Chapter 5). However, in order to establish that FN is also involved in early fish development, we first performed a pilot study in which the functional role of FN was blocked by treatment with GRGDS, interfering with interactions between FN and its receptor. Since this treatment resulted both in retardation of epiboly, and a lack of involution, it became interesting to study the localization of FN during respective stages. Using immunocytological methods, FN first appeared present on a number of cell membranes during early epiboly. During the progress of gastrulation (from 50% epiboly onwards) we observed a gradually increasing number of epiblastcells, which were labeled on their YSL facing surface. They were first present in front of the migratory hypoblast cells and later partly separated the hypoblast cells from the epiblast. The results suggest that also in cyprinid fishes FN may be involved in the guidance of gastrulating cells.In conclusion, PGCs in embryos of cyprinid fishes can be recognized from late gastrula stages onwards by the presence of nuage. They are supposed to arise epigenetically from the mesoderm, possibly by inducing influence of endodermal cells. Determination processes of both PGCs and somatic cells probably do not start until early gastrulation (50% epiboly). FN may be involved directly (induction processes) or indirectly (morphogenetic movements resulting in induction) in epigenetic processes